The purpose of Project 3 is to determine the role of the TIM-1 protein in the development of Th2-biased inflammation and asthma. Tim1 is a gene that we recently discovered using a unique congenic mouse model, which regulates Th2 responsiveness and the development of asthma. The human homologue of Tim1 lies on chromosome 5q33.2, a region repeatedly linked to asthma. Moreover, the human Tim1 encodes the cellular receptor for the hepatitis A virus (HAV), suggesting that CD4 T cells and TIM-1 may mediate the known protective effect of prior infection with HAV on the development of atopy. Since the prevalence of HAV infection is greatly reduced in industrialized countries, our findings may explain in part the enormous increase in asthma prevalence occurring over the past two decades, and strongly suggest that Tim1 plays a major role in directly regulating the development of human asthma and allergy. In Specific Aim 1, we will determine what murine cell types express TIMs, and the kinetics of this expression using anti-TIM mAb and Northern analysis. We believe that TIMs critically regulate T cell differentiation, as evidenced by the fact that TIM1 is preferentially expressed on Th2 cells, and TIM3 is preferentially expressed on Th1 cells. Thus, by identifying when and where TIMs are expressed we will obtain essential information about, and clues regarding, TIM function. In Specific Aim 2, we will utilize Tim1 knockout mice and TIM blocking reagents (fusion proteins and mAb) to directly study the precise role of TIM-1 and TIM-3 in the development of Th2 responses and allergen-induced airway hyperreactivity. Dr. Arlene Sharpe at Harvard University will provide assistance in making Tim1 knockout mice, using theCre/Iox P system and embryonic stem cells derived from BALB/c mice. In Specific Aim 3, we will investigate signaling pathways of murine TIM-1, whose intracellular sequence includes an Itk phosphorylation site. We will determine how TIM-1 intracellular signaling fits in with Itk, which is known to play an essential role in the development of Th2 polarized responses. In addition, we will determine how the BALB/c and HBA TIM-1 alleles differ with regard to intracellular signaling events. These studies will greatly increase our understanding of the function of TIMs in the regulation of Th2 responses and allergen induced airway hyperreactivity. These studies will thereby characterize a crucial regulator of CD4 T cell differentiation and a novel and extremely important asthma susceptibility gene that we have recently discovered.